CLONING AND CHARACTERIZATION OF THE GENE FOR THE SOMATIC FORM OF DNA TOPOISOMERASE-I FROM XENOPUS-LAEVIS

Two distinct tissue-specific forms of DNA topoisomerase I with M(r) of 165 and 110 kDa have been purified from oocytes and somatic cells res pectively of the African frog Xenopus laevis. In this paper, cDNAs enc oding a Xenopus topoisomerase I were cloned using PCR primers derived from sequences of yeast and human topoisomerase I. A polypeptide expre ssed from a portion of the coding sequence was recognized by an antise rum directed against the somatic topoisomerase I that had previously b een shown to be unable to cross-react with the oocyte enzyme. Thus, th e clone encodes the somatic cell topoisomerase I. An antiserum raised against a synthetic peptide containing the sequence surrounding the ac tive site tyrosine of the somatic topoisomerase I reacts with the enzy mes purified from both oocytes and somatic cells, indicating that the two enzymes share some limited sequence homology. RNA blot hybridizati on showed that oocytes contain an abundant store of somatic topoisomer ase I mRNA that is not efficiently polyadenylated in oocytes. This sto red RNA contains a consensus cytoplasmic polyadenylation element that is found in a variety of mRNAs that are translationally repressed in o ocytes. Microinjection into oocytes of in vitro transcribed mRNA prepa red from a Myc-tagged construct of the somatic topoisomerase I sequenc e is translated to yield a 110 kDa product. This suggests that the ooc yte-specific 165 kDa topoisomerase I is not produced by tissue-specifi c post-translational modification of the somatic topoisomerase I. The oocyte enzyme appears to be produced from a minor mRNA species in oocy tes that has not yet been identified.